Method of detecting colon cancer marker

ABSTRACT

It is intended to provide a non-invasive and convenient method of detecting a tumor marker for diagnosing colon cancer which is superior in sensitivity and specificity to the existing fecal occult blood test. More specifically speaking, a method of detecting a tumor marker for diagnosing colon cancer which comprises collecting biological sample which is immediately frozen using liquid nitrogen in some cases, homogenizing the sample in the presence of an inhibitor of an RNA digesting enzyme to give a suspension, extracting RNA from the obtained suspension, subjecting the extracted RNA to reverse transcription to give cDNA. amplifying the obtained cDNA and then detecting the thus amplified cDNA. This method is characterized by involving no procedure of separating cell components from the biological sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/549,389, filed Sep. 14, 2005, which is the United States NationalPhase Application of International Application PCT/JP2003/11972, filedon Sep. 19, 2003, which is based upon and claims the benefit of priorityfrom Japanese Patent Application No. 2003-75552, filed Mar. 19, 2003,the entire contents of all which are incorporated herein by reference.

The present invention relates to a tumor marker detecting method fordiagnosing colon cancer comprising a process of extracting RNA from abiological sample, characterized by involving no procedure of separatingcell components from the biological sample.

BACKGROUND OF THE INVENTION

The deaths by colon cancer are increasing. The number of deaths by coloncancer is the fourth large among male, and the second large among femaledeaths in all cancer deaths (Statistics of Japanese cancer deaths in1999). According to an estimation of cancer patients in 2015 in Japan,number of colon cancer patients is estimated to be the first in bothmale and female. Global measures to counter colon cancer includingsecondary prevention are thus required, and mass screening of cancer maybe one of the most effective methods.

For the mass screening of cancer, it is important that the detectionmethod is easy and non-invasive. The only non-invasive method nowavailable is the method to examine existence of occult blood in feces,that is, the fecal occult blood test, and is used extensively as astandard method of the mass screening of colon cancer.

However, the fecal occult blood test has rather low 30 sensitivity andspecificity (the sensitivity: 30 to 90%, the specificity: 70 to 98%),because appearance of hemoglobin in feces is not specific to tumor.Therefore, there is a shortcoming that quite a few false negatives andfalse positives exist.

Also, in the diagnosis of colon cancer, after or in parallel with thescreening by the immunological fecal occult blood test, totalcolonoscopy or a combination of Ba-enema and sigmoidoscopy has beenadopted. There is thus a shortcoming that it needs much time and effort.

As alternative methods to the fecal occult blood test, methods using DNAare reported, such as detection of mutations in K-ras, p-53, or APCgenes, or detection of microsatellite instability in feces (D.Sidransky, et al., Science, 256. Apr. 3, 1992, 102-105; S. M. Dong, etal., Journal of the National Cancer Institute. 93 (11). Jun. 11, 2001,858-865; G. Traverso, et al., The New England Journal of Medicine, 346(5), Jan. 31, 2002, 311-320; G. Traverso, et al., The Lancet, 359, Feb.2, 2002, 403-404).

These methods using DNA are non-invasive and can capture the directchanges in cancer cells, and have characteristics of having highspecificity, and so are considered to be a hopeful method in the future.However, it has a demerit that the sensitivity is lower compared to thefecal occult blood test, a prior art, and is rather time andeffort-consuming.

Further, as an alternative method to the fecal occult blood test, inorder to detect gene expression more directly, a method for detectingmRNA of protein kinase C (PKC) or the like in the feces has beendeveloped (L. A. Davidson, et al., Carcinogenesis, 19(2), 1998, 253-257;R. J. Alexander and R. F. Raicht, Digestive Diseases and Sciences,43(12), 1998, 2652-2658; T. Yamao, et al., Gastroenterology, 114(6),1998, 1196-1205).

However, the method making use of RNA described above could not have thesensitivity exceeding that of the fecal occult blood test method,because it was impossible to extract RNA easily and efficiently from asmall amount of feces.

A method to detect RNA qualitatively and quantitatively by combining thePCR method with the reverse transcriptase reaction (RT), has been known.This RT-PCR method is superior to Northern blot technique in the highsensitivity to be able to detect trace molecules, and is moreadvantageous than the in situ hybridization technique in speed andeasiness of manipulation.

However, since RNA is more unstable compared with DNA and is alwayssubjected to a danger of decomposition by RNA digesting enzymes (RNases)which are ubiquitous in all the biological samples and very stable,strict control to avoid contamination of RNases is necessary in theRT-PCR method, during and after purification processes of RNA.

Therefore, when RNA is extracted from the feces, which is a biologicallyvery crude sample, a process to separate the cell fraction in advancehas been necessary, to exclude effects of RNases.

Accordingly, it has been considered impossible to extract RNA directlyfrom feces containing enormous amount of RNases derived from very largeamount of microorganisms, and, a separation of the cell fraction hasbeen considered to be essential for removing at least exogenous RNasesderived from microorganisms or the like.

Surprisingly, however, the inventor of the present invention found thatin some cases, homogenization of frozen biological materials in thepresence of RNase inhibitors can resolve the problems described above,and has completed the present invention.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide anon-invasive and convenient tumor marker detecting method for diagnosingcolon cancer, which is superior in sensitivity and specificity to theexisting fecal occult blood test.

The present invention is a method to prepare a sample for extracting RNAused to detect a tumor marker for diagnosing colon cancer, comprisingfollowing process;

a) a process to homogenize the collected biological sample in thepresence of an RNase inhibitor to prepare a suspension thereof;characterized by involving no procedure of separating cell componentsfrom the biological sample.

Here, said collected biological sample is preferably frozen.

Moreover the present invention is the method described above, whereinthe RNase inhibitor is guanidine thiocyanate.

Also, the present invention is the method described above, wherein thebiological sample is feces.

Further, the present invention is a tumor marker detecting method fordiagnosing colon cancer, comprising the following processes in additionto the process described above:

b) a process to extract RNA from the obtained sample for extracting RNA;

c) a process to reverse transcribe the extracted RNA to give cDNA;

d) a process to amplify the obtained cDNA; and,

e) a process to detect the amplified cDNA.

The present invention is a tumor marker detecting method for diagnosingcolon cancer, wherein said tumor marker is COX-2.

The present invention is also a kit for preparing a sample to extractRNA used in the tumor marker detecting method for diagnosing coloncancer, comprising the following means;

a) a means to homogenize the collected biological sample in the presenceof an RNase inhibitor, to prepare a suspension thereof; said kit beingcharacterized by involving no means of separating cell components fromthe biological sample.

Also, the kit of the present invention preferably contains a means tofreeze said collected biological sample.

The present invention is the kit described above, wherein the RNaseinhibitor is guanidine thiocyanate.

The present invention is also the kit described above, wherein thebiological sample is feces.

Moreover, the present invention is a tumor marker detecting kit fordiagnosing colon cancer, comprising the following means:

b) a means to extract RNA from the obtained sample for extracting RNA;

c) a means to reverse transcribe the extracted RNA to give cDNA;

d) a means to amplify the obtained cDNA; and,

e) a means to detect the amplified cDNA. Further, the present inventionis the kit described above, wherein said tumor marker is COX-2.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows' a result of an electrophoresis in Example 2. The lane 1shows total RNA extracted from human feces with the method by Alexanderet al. The lane 2 shows total RNA extracted from human feces with themethod of the present invention. The lane 3 shows total RNA extractedfrom a human colon cancer tissue. The lane M shows the molecular weightmarkers.

THE BEST MODE FOR CARRYING OUT THE INVENTION

As for the RNase inhibitors of the present invention, guanidinethiocyanate, Isogene, (a homogenous liquid containing phenol andguanidine thiocyanate), Ultraspec II (a registered trade mark) (a 14Msolution of guanidine salts, urea and a RNA binding resin) and the likeare included.

The biological samples of the present invention are tissues of animalsand plants, body fluids, excrements and the like, and preferably arefeces, and more preferably are human feces.

The biological samples of the present invention can be used as they are,or. in some cases, after frozen.

Freezing methods can be any conventional methods, and preferably amethod using liquid nitrogen. The freezing (and preserving) temperatureis −1 to −196° C., preferably −20 to −196° C., preferably −75 to −196°C., more preferably −110 to −196° C. and most preferably −196° C.

The frozen sample may be preserved in frozen state. The preservationtemperature is −75 to −196° C., preferably −110 to −196° C., and morepreferably −196° C. The preservation period is one day to 10 years,preferably one day to 3 years, and more preferably one day to one year.

The tumor marker used in the present invention is COX-2,matrixmetalloprotease (MMP), c-met, CD44 variants, EGF-R, EF-1, Wnt-2,Bradeion, SKP2, KPC-1, KPC-2, PRL-3, Angiogenin, Integrin, Snail,Dysadherin, or the like, and is preferably COX-2.

The processes c) to e) described above are called as the RT-PCR method,and can be carried out, for example, according to the description by T.Sekiya et al. eds., Forefront of PCR method, 1997, Kyoritu Pub.,187-196.

Extraction of RNA from the suspension can be carried out using methodswell known in the art, and using commercially available kits, forexample, RNeasy Mini (QIAGEN) or RNA Extraction Kit (Pharmacia Biotech).

“Reverse transcription” in the present invention means conversion of RNAto the complementary DNA (cDNA) using a reverse transcriptase. Thereverse transcription reaction is usually conducted using a solutioncontaining a buffer, salts such as MgCl₂, KCl, and the like,dithiothreitol (DTT), a primer, kinds of deoxyribonucleotides, RNaseinhibitors, and a reverse transcriptase. The salts described above canbe appropriately replaced by other salts after testing. Proteins such asgelatine, albumin or the like, or detergents can also be added.

For amplification of cDNA carried out subsequent to the reversetranscription, the PCR technique is usually adopted. The PCR reactionmixture usually contains a buffer, salts such as MgCl₂ and KCl, primers,kinds of deoxyribonucleotides, and a heat resistant polymerase. Thesalts described above can be appropriately replaced by other salts aftertesting. Proteins such as gelatine, albumin or the like,dimethylsulphoxide, detergents, or the like, can also be added.

For amplification of cDNA, the LAMP method (Japan Patent No. 3313358) orthe ICAN method (Japan Patent Laid-Open No. 2001-136965) can be used.

A “primer” in the present invention means an oligonucleotide which worksas a synthesis initiation point in the case of cDNA synthesis orpolynucleotide amplification. The primer is preferably a single-strand,but a double-strand can also be used. When the primer is adouble-strand, it is preferable to make it single-stranded prior to theamplification reaction. The primer can be synthesized according to amethod well known in the art, or can be isolated from the livingorganisms.

The reverse transcriptase used in the reverse transcription reaction isan enzyme capable of reverse transcribing RNA to cDNA. As for thereverse transcriptase, there are reverse transcriptases derived fromretroviruses such as RAV (Rous associated virus), AMV (Avianmyeloblastosis virus) and the like, and reverse transcriptases derivedfrom mouse retroviruses such as MMLV (Moloney murine leukemia virus) andthe like, but it is not limited to the aboves.

As the heat resistant polymerase used for PCR, Taq polymerase can benominated, but it is not confined to this.

As the detection method of amplified DNA, electrophoresis using agarosegel can be used, but the method may not be confined to this.

Further, the kit according to the present invention may contain aninstruction describing the methods of the present invention.

Example 1

The following examples illustrate the present invention, but do notlimit the invention.

Among patients hospitalized in the First Department of Internal Medicineof Hamamatsu University School of Medicine for detailed examination andtherapy, 30 cases confirmed to have colon cancer and 22 cases to have notumor or inflammatory alteration in their colon (non colon disorder) bythe total colonoscopy were selected as the subject of the study.Informed consents of all the cases had been obtained.

As soon as possible after sampling feces, the feces were separated into5 ml tubes about 1 g each, were frozen using liquid nitrogen, and werestored at −80° C. Also, for comparison and reference, human hemoglobin(Hb) in the feces of each sample was measured by the immunological fecaloccult blood test. Tissue biopsy specimen, taken both from the cancerand the normal parts when the endoscopy was carried out before thetherapy, were frozen by liquid nitrogen and stored at −80° C. Then,feces were homogenized using a homogenizer, guanidine salt, and phenol,and whole RNA was extracted using chloroform and ethanol.

One μg of the obtained RNA was reverse transcribed using ReverScript II(a registered trade mark), (reaction mixture volume; 20 μl, Wako PureChemical Industries) to give cDNA. A part thereof was amplified by meansof nested PCR using GeneTaq (Wako). The PCR product obtained waselectrophresed on 4% agarose gel, and stained by ethidium bromide.

Here, the primers used were: the random primers in reversetranscription, and in PCR, were those reported by Gerhard (JJCO, 1994)for CEA, and were originally designed for COX-2. The first round of PCRwas executed 20 cycles, and the second round 25 cycles.

The followings indicate the primers used.

<CEA>

(SEQ ID NO: 1) Forward 1:  5′-TCTGGAACTTCTCCTGGTCTCTCAGCTGG-3′(SEQ ID NO: 2) Forward 2: 5′-GGGCCACTGCTGGCATCATGATTG-3′ (SEQ ID NO: 3)Reverse:  5′-TGTAGCTGTTGCAAATGCTTTAAGGAAGAAGC3′

<COX-2>

(SEQ ID NO: 4) Forward 1: 5′-CTGAAAACTCCAAACACAG-3′ (SEQ ID NO: 5)Forward 2: 5′-GCACTACATACTTACCCACTTCAA-3′ (SEQ ID NO: 6) Reverse: 5′-ATAGGAGAGGTTAGAGAAGGCT-3′

Results

Feces from 30 colon cancer cases (3 early cancer and 27 advanced cancercases) and from 22 cases in the control group were examined by RT-PCR,in order to detect CEA and COX-2, and the following results wereobtained.

CEA was detected in all cases among the 30 colon cancer cases, and in 21among 22 cases in the control group. Also, it turned out that RNAsuitable for RT-PCR amplification could be extracted from both samples.

COX-2 was detected in 27 cases among the 30 colon cancer cases (caeca:2/2, ascending colon: ⅗, descending colon: 1/1, sigmoid colon: 7/7,rectum: 12/13; early cancer: ⅔, advanced cancer: 25/27), but was notdetected in any of 22 cases in the control group (sensitivity: 90%,specificity: 100%).

In the immunological fecal occult blood test, 23 among 28 colon cancercases and 3 among 22 control cases were positive (sensitivity: 82.1%,specificity: 86.3%).

Among three COX-2 negative colon cancer cases, one was positive in theimmunological fecal occult blood test, and 2 were negative.

COX-2 was detected in 3 among 5 colon cancer cases negative to theimmunological fecal occult blood test,

Example 2

The amount-and the distribution of molecular weights of total RNAobtained from human feces according to the method of the presentinvention were compared with those obtained according to Alexander'smethod (R. J. Alexander and R. F. Raicht, Digestive Diseases andSciences, 43(12), 1998, 2652-2658). As a control, total RNA wasextracted from the human colon cancer tissues using a commerciallyavailable RNA extraction reagent (ISOGEN, Wako)

The same amount of total RNA extracted from each sample waselectrophoresed on an agarose gel.

Two main bands recognized on the lane 3 (RNA derived from human coloncancer tissues) show 28s and 18s rRNAs. Smeared parts thereon indicatethat various kinds of high molecular weight RNAs are contained in theobtained total RNA.

Two main bands recognized on the lane 2 (RNA derived from feces obtainedby the method of the present invention) show 23s and 16s rRNAs derivedfrom enteric bacteria. Since smeared parts were also recognized thereonsimilarly to the lane 3, the total RNA obtained from the feces by themethod of the present invention is considered to contain also variouskinds of high molecular weight RNAs.

Contrarily, any bands and smears were not detected at all in the lane 1,showing that high molecular weight RNAs were not contained in theextract of the sample.

In fact, the desired products were obtained from the sample of lane 2 bythe RT-PCR technique, but no PCR products were obtained from the sampleof lane 1.

From the results of the present studies, it became obvious that the RNAextracted from the human feces by the method of the present inventioncan be amplified by means of the RT-PCR technique. Also, the detectionof COX-2 from the feces by the RT-PCR technique had 90% sensitivity and100% specificity, and it is proved that the present invention issuperior to a conventional technique of the immunological fecal occultblood test.

Further, since the method of the present invention needs smaller amountof feces for detection and has higher detection sensitivity compared tothe detection of the gene mutation of APC, K-ras, or p53, it can savelargely the time and effort needed for the detection.

While the conventional technique of the fecal occult blood test targetsa general and indirect event, “bleeding” from the lesion, the method ofthe present invention targets a specific and direct event, theexpression of a marker of carcinogenesis, COX-2. Therefore, the dataobtained by the method of the present invention provide diagnosis withhigher quality.

Accordingly, the method of the present invention is clinically veryuseful as a novel non-invasive screening method with high specificityand high sensitivity.

1. A method, comprising: a) homogenizing collected feces in the presenceof an RNase inhibitor to prepare a suspension thereof, withoutseparating cell components from the feces; and b) extracting total RNAfrom the suspension from step a) to provide extracted total RNA.
 2. Themethod of claim 1, further comprising: c) carrying out reversetranscription on the extracted total RNA from step b) to provide cDNA;d) amplifying the cDNA from step c); and e) detecting the amplified cDNAfrom step d).
 3. The method according to claim 1, wherein the feces isfrozen.
 4. The method according to claim 1, wherein the feces is humanfeces.
 5. The method according to claim 1, wherein said RNase inhibitoris a guanidine salt.
 6. The method according to claim 2, wherein thefeces is frozen.
 7. The method according to claim 2, wherein the fecesis human feces.
 8. The method according to claim 2, wherein said RNaseinhibitor is a guanidine salt.